Unraveling the structure of DNA during overstretching by using multicolor, single-molecule fluorescence imaging

Abstract

Single-molecule manipulation studies have revealed that doublestranded DNA undergoes a structural transition when subjected to tension. At forces that depend on the attachment geometry of the DNA (65 pN or 110 pN), it elongates Ϸ1.7-fold and its elastic properties change dramatically. The nature of this overstretched DNA has been under debate. In one model, the DNA cooperatively unwinds, while base pairing remains intact. In a competing model, the hydrogen bonds between base pairs break and two single DNA strands are formed, comparable to thermal DNA melting. Here, we resolve the structural basis of DNA overstretching using a combination of fluorescence microscopy, optical tweezers, and microfluidics. In DNA molecules undergoing the transition, we visualize double-and single-stranded segments using specific fluorescent labels. Our data directly demonstrate that overstretching comprises a gradual conversion from double-stranded to singlestranded DNA, irrespective of the attachment geometry. We found that these conversions favorably initiate from nicks or free DNA ends. These discontinuities in the phosphodiester backbone serve as energetically favorable nucleation points for melting. When both DNA strands are intact and no nicks or free ends are present, the overstretching force increases from 65 to 110 pN and melting initiates throughout the molecule, comparable to thermal melting. These results provide unique insights in the thermodynamics of DNA and DNA-protein interactions.DNA melting ͉ fluorescence microscopy ͉ optical trapping ͉ single-molecule techniques ͉ single-stranded DNA

Peeling back the mystery of DNA overstretching

“…The structure of the DNA during these transitions is difficult to measure with traditional structural methods because it only occurs on a single molecule. More than a decade after its discovery, van Mameren et al (1) have, in this issue of PNAS, directly visualized the mechanics of DNA overstretching for the first time by combining single-molecule force spectroscopy and fluorescence imaging. They show that, regardless of whether or not the molecule can freely rotate, when DNA is stretched to high force it will melt, as the work done by the force to increase the length of the DNA converts double-stranded duplex DNA into ssDNA.…”

“…By establishing force-induced DNA melting as the mechanism by which DNA undergoes conformational transitions under force, van Mameren et al (1) have significantly advanced the field of singlemolecule DNA stretching. Based on these results, DNA stretching studies with or without DNA binding ligands must be interpreted in terms of DNA melting.…”

“…Thus, despite many years of detailed study, DNA stretching experiments lacked See companion article on page 18231. 1 To whom correspondence should be addressed. E-mail: mark@neu.edu.…”

Stretching single DNA molecules to demonstrate high‐force capabilities of holographic optical tweezers

“…The forceextension curve is highly nonlinear, conforming at low forces to the entropic worm-like chain model of polymer elasticity and exhibiting a plateau at a force of 65 pN. In this so-called overstretch plateau, the molecular contour length increases by 70% as the two strands of DNA melt [15]. Observation of a plateau at 65 pN is a clear signature of a single, torsionally unconstrained dsDNA.…”

Optical tweezers studies of transcription by eukaryotic RNA polymerases

“…In combination with fluorescence microscopy, optical tweezers were used in DNA overstretching experiments and for determining DNA-protein interactions (88)(89)(90). Angular optical trapping (AOT) was developed for simultaneous detection of force and torque (91,92), which enabled studying transcription dynamics and nucleosome stability under conditions of applied torque on the DNA (93).…”